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Abstract The genetic architecture of Parkinson’s disease (PD) is complex and multiple brain cell subtypes are involved in the neuropathological progression of the disease. Here we aimed to advance our understanding of PD genetic complexity at a cell subtype precision level. Using parallel single-nucleus (sn)RNA-seq and snATAC-seq analyses we simultaneously profiled the transcriptomic and chromatin accessibility landscapes in temporal cortex tissues from 12 PD compared to 12 control subjects at a granular single cell resolution. An integrative bioinformatic pipeline was developed and applied for the analyses of these snMulti-omics datasets. The results identified a subpopulation of cortical glutamatergic excitatory neurons with remarkably altered gene expression in PD, including differentially-expressed genes within PD risk loci identified in genome-wide association studies (GWAS). This was the only neuronal subtype showing significant and robust overexpression of SNCA. Further characterization of this neuronal-subpopulation showed upregulation of specific pathways related to axon guidance, neurite outgrowth and post-synaptic structure, and downregulated pathways involved in presynaptic organization and calcium response. Additionally, we characterized the roles of three molecular mechanisms in governing PD-associated cell subtype-specific dysregulation of gene expression: (1) changes in cis-regulatory element accessibility to transcriptional machinery; (2) changes in the abundance of master transcriptional regulators, including YY1, SP3, and KLF16; (3) candidate regulatory variants in high linkage disequilibrium with PD-GWAS genomic variants impacting transcription factor binding affinities. To our knowledge, this study is the first and the most comprehensive interrogation of the multi-omics landscape of PD at a cell-subtype resolution. Our findings provide new insights into a precise glutamatergic neuronal cell subtype, causal genes, and non-coding regulatory variants underlying the neuropathological progression of PD, paving the way for the development of cell- and gene-targeted therapeutics to halt disease progression as well as genetic biomarkers for early preclinical diagnosis.

Abstract Background The genetic underpinnings of late-onset Alzheimer’s disease (LOAD) are yet to be fully elucidated. Although numerous LOAD-associated loci have been discovered, the causal variants and their target genes remain largely unknown. Since the brain is composed of heterogenous cell subtypes, it is imperative to study the brain on a cell subtype specific level to explore the biological processes underlying LOAD. Methods Here, we present the largest parallel single-nucleus (sn) multi-omics study to simultaneously profile gene expression (snRNA-seq) and chromatin accessibility (snATAC-seq) to date, using nuclei from 12 normal and 12 LOAD brains. We identified cell subtype clusters based on gene expression and chromatin accessibility profiles and characterized cell subtype-specific LOAD-associated differentially expressed genes (DEGs), differentially accessible peaks (DAPs) and cis co-accessibility networks (CCANs). Results Integrative analysis defined disease-relevant CCANs in multiple cell subtypes and discovered LOAD-associated cell subtype-specific candidate cis regulatory elements (cCREs), their candidate target genes, and trans-interacting transcription factors (TFs), some of which, including ELK1, JUN, and SMAD4 in excitatory neurons, were also LOAD-DEGs. Finally, we focused on a subset of cell subtype-specific CCANs that overlap known LOAD-GWAS regions and catalogued putative functional SNPs changing the affinities of TF motifs within LOAD-cCREs linked to LOAD-DEGs, including APOE and MYO1E in a specific subtype of microglia and BIN1 in a subpopulation of oligodendrocytes. Conclusions To our knowledge, this study represents the most comprehensive systematic interrogation to date of regulatory networks and the impact of genetic variants on gene dysregulation in LOAD at a cell subtype resolution. Our findings reveal crosstalk between epigenetic, genomic, and transcriptomic determinants of LOAD pathogenesis and define catalogues of candidate genes, cCREs, and variants involved in LOAD genetic etiology and the cell subtypes in which they act to exert their pathogenic effects. Overall, these results suggest that cell subtype-specific cis–trans interactions between regulatory elements and TFs, and the genes dysregulated by these networks contribute to the development of LOAD.

ABSTRACT Protein kinase A (PKA) signaling plays a critical role in the growth and development of all eukaryotic microbes. However, few direct targets have been characterized in any organism. The fungus Aspergillus fumigatus is a leading infectious cause of death in immunocompromised patients, but the specific molecular mechanisms responsible for its pathogenesis are poorly understood. We used this important pathogen as a platform for a comprehensive and multifaceted interrogation of both the PKA-dependent whole proteome and phosphoproteome in order to elucidate the mechanisms through which PKA signaling regulates invasive microbial disease. Employing advanced quantitative whole-proteomic and phosphoproteomic approaches with two complementary phosphopeptide enrichment strategies, coupled to an independent PKA interactome analysis, we defined distinct PKA-regulated pathways and identified novel direct PKA targets contributing to pathogenesis. We discovered three previously uncharacterized virulence-associated PKA effectors, including an autophagy-related protein, Atg24; a CCAAT-binding transcriptional regulator, HapB; and a CCR4-NOT complex-associated ubiquitin ligase, Not4. Targeted mutagenesis, combined with in vitro kinase assays, multiple murine infection models, structural modeling, and molecular dynamics simulations, was employed to characterize the roles of these new PKA targets in growth, environmental and antimicrobial stress responses, and pathogenesis in a mammalian system. We also elucidated the molecular mechanisms of PKA regulation for these effectors by defining the functionality of phosphorylation at specific PKA target sites. We have comprehensively characterized the PKA-dependent phosphoproteome and validated PKA targets as direct regulators of infectious disease for the first time in any pathogen, providing new insights into PKA signaling and control over microbial pathogenesis. IMPORTANCE PKA is essential for the virulence of eukaryotic human pathogens. Understanding PKA signaling mechanisms is therefore fundamental to deciphering pathogenesis and developing novel therapies. Despite its ubiquitous necessity, specific PKA effectors underlying microbial disease remain unknown. To address this fundamental knowledge gap, we examined the whole-proteomic and phosphoproteomic impacts of PKA on the deadly fungal pathogen Aspergillus fumigatus to uncover novel PKA targets controlling growth and virulence. We also defined the functional consequences of specific posttranslational modifications of these target proteins to characterize the molecular mechanisms of pathogenic effector regulation by PKA. This study constitutes the most comprehensive analysis of the PKA-dependent phosphoproteome of any human pathogen and proposes new and complex roles played by PKA signaling networks in governing infectious disease.

Cellular recycling via autophagy-associated proteins is a key catabolic pathway critical to invasive fungal pathogen growth and virulence in the nutrient-limited host environment. Protein kinase A (PKA) is vital for the growth and virulence of numerous fungal pathogens. However, the underlying basis for its regulation of pathogenesis remains poorly understood in any species. Our Aspergillus fumigatus PKA-dependent whole proteome and phosphoproteome studies employing advanced mass spectroscopic approaches identified numerous previously undefined PKA-regulated proteins in catabolic pathways. Here, we demonstrate reciprocal inhibition of autophagy and PKA activity, and identify 16 autophagy-associated proteins as likely novel PKA-regulated effectors. We characterize the novel PKA-phosphoregulated sorting nexin Atg20, and demonstrate its importance for growth, cell wall stress response, and virulence of A. fumigatus in a murine infection model. Additionally, we identify physical and functional interaction of Atg20 with previously characterized sorting nexin Atg24. Furthermore, we demonstrate the importance of additional uncharacterized PKA-regulated putative autophagy-associated proteins to hyphal growth. Our data presented here indicate that PKA regulates the autophagy pathway much more extensively than previously known, including targeting of novel effector proteins with fungal-specific functions important for invasive disease.

ABSTRACT Invasive aspergillosis (IA), caused by the filamentous fungal pathogen Aspergillus fumigatus, is a major cause of death among immunocompromised patients. The cyclic AMP/protein kinase A (PKA) signaling pathway is essential for hyphal growth and virulence of A. fumigatus, but the mechanism of regulation of PKA remains largely unknown. Here, we discovered a novel mechanism for the regulation of PKA activity in A. fumigatus via phosphorylation of key residues within the major catalytic subunit, PkaC1. Phosphopeptide enrichment and tandem mass spectrometry revealed the phosphorylation of PkaC1 at four sites (S175, T331, T333, and T337) with implications for important and diverse roles in the regulation of A. fumigatus PKA. While the phosphorylation at one of the residues (T333) is conserved in other species, the identification of three other residues represents previously unknown PKA phosphoregulation in A. fumigatus. Site-directed mutagenesis of the phosphorylated residues to mimic or prevent phosphorylation revealed dramatic effects on kinase activity, growth, conidiation, cell wall stress response, and virulence in both invertebrate and murine infection models. Three-dimensional structural modeling of A. fumigatus PkaC1 substantiated the positive or negative regulatory roles for specific residues. Suppression of PKA activity also led to downregulation of PkaC1 protein levels in an apparent novel negative-feedback mechanism. Taken together, we propose a model in which PkaC1 phosphorylation both positively and negatively modulates its activity. These findings pave the way for future discovery of fungus-specific aspects of this key signaling network. IMPORTANCE Our understanding of signal transduction networks in pathogenic fungi is limited, despite the increase in invasive fungal infections and rising mortality rates in the immunosuppressed patient population. Because PKA is known to be essential for hyphal growth and virulence of A. fumigatus, we sought to identify fungus-specific regulatory mechanisms governing PKA activity. In this study, we identify, for the first time, a novel mechanism for the regulation of PKA signaling in which differential phosphorylation of the PkaC1 catalytic subunit can lead to either positive or negative regulation of activity. Furthermore, we show that inactivation of PKA signaling leads to downregulation of catalytic subunit protein levels in a negative-feedback mechanism distinct from expression patterns previously reported in the yeasts. Our findings represent a divergence in the regulation of PKA signaling in A. fumigatus, which could potentially be exploited as a target and also open the avenue for discovery of fungus-specific downstream effectors of PKA.